The present invention relates to modified microorganisms suitable for use as live vaccines. The present invention also relates to the use of modified microorganisms as biological vectors. The present invention further relates to vaccine compositions.
Bovine respiratory disease (BRD) complex, shipping fever, or pneumonic pasteurellosis, is a multifactorial disease whereby a combination of viral infection, adverse environment and poor immune status may combine to predispose animals to bacterial infections. BRD is a major cause of economic loss in the cattle feed lot industry. The principal microorganism associated with the disease is the bacteria Pasteurella haemolytica serotype 1. (Schiefer, et al., 1978). Under normal conditions P. haemolytica is a component of the normal flora of the upper respiratory tract, it is only when pulmonary clearance mechanisms are impaired that colonisation of the lung occurs resulting in disease (Frank and Smith, 1983). A number of virulence factors have been associated with P. haemolytica, including surface structures such as the capsular polysaccharide (Adam et al. 1984), and a secreted exotoxin which is heat labile and specific for ruminant leukocytes (Shewen and Wilkie, 1982).
The exotoxin, or leukotoxin (Lkt), may contribute to pathogenesis by impairing the primary lung defenses and subsequent immune responses or by causing inflammation as a result of leukocyte lysis. Characterisation of the Lkt has shown it to be a member of the RTX family of toxins (Strathdee and Lo, 1989) which are produced by a variety of bacteria including Actinobacillus spp, Proteus vulgaris, Morganella morganii, Bordetella pertussis, and the most characterised produced by E. coli. All RTX toxins function by producing pores in the target cells, thereby interrupting osmotic balance, leading to rupture of the target cell. Although the mode of action is identical for RTX toxins their target cells vary greatly in type and cross-species specificity. Structurally, this family of toxins are characterised by the presence of glycine rich repeat structures within the toxin that bind to calcium and may have a role in target cell recognition and binding, a region of hydrophobic domains that are involved in pore formation, the requirement for post translational activation, and dependence on a C-terminal signal sequence for secretion. Production and secretion of an active RTX toxin requires the activity of at least four genes, C, A, B, and D. The A gene encodes the structural toxin, the C gene encodes a post-translational activator and the B and D genes encode proteins that are required for secretion of the active toxin. The Lkt is encoded by an operon that consists of the four contiguous genes (CABD), transcribed by a single promoter. The Lkt differs from a number of other RTX toxins, which have a broad host cell specificity, by having a target cell specificity restricted to ruminant leukocytes (Reviewed: Coote, 1992).
The Lkt has also been associated with protective immunity; with anti-toxin antibodies in the field relating to disease resistance, and a commercial culture supernatant vaccine (Presponse; Langford Inc., Guelph, Ontario, Canada) containing Lkt showing efficacy in reducing the incidence and severity of pneumonia following experimental challenge and in the feed lot (Gentry et al., 1985; Mosier et al., 1986; 1989; Shewen and wlkie, 1987; Shewen et al., 1988). This culture supernatant vaccine, in addition to inducing anti-Lkt antibodies, also stimulates an immune response to other soluble antigens present in the culture supernatant, and therefore a direct correlation between anti-Lkt and protection can not be claimed.
The use of Pasteurella bacterins (inactive vaccines) in the field has had limited success in controlling pneumonic pasteurellosis, in several field trials the administration of bacterin based vaccine has not protected against disease or in some cases had led to an enhancement of disease (Bennett, 1982; Morter et al., 1982). Bacterin vaccines also have the disadvantages of requiring the use of adjuvants, may result in site reactions, and in a number of cases require multiple dose to obtain protection.
It is an object of the present invention to alleviate one or more of the problems of the prior art.
Accordingly, in one aspect the present invention provides a modified microorganism which produces an Lkt toxin, wherein said Lkt toxin is partially or fully inactivated.
The term xe2x80x9cmodifiedxe2x80x9d includes modification by recombinant DNA techniques or other techniques such as chemical- or radiation-induced mutagenesis. Where recombinant DNA techniques involve the introduction of foreign DNA into host cells, the DNA may be introduced by any suitable method. Suitable methods include transformation of competent cells, transduction, conjugation and electroporation.
In a further embodiment of the present invention, there is provided a modified microorganism wherein an Lkt toxin operon including an Lkt structural gene and/or a post translational activator of the organism is partially or fully inactivated.
The term xe2x80x9cLkt toxin operonxe2x80x9d as used herein the claims and description is intended to include those genes involved in the expression of an Lkt toxin being a product of the Lkt toxin operon. The genes included in the Lkt toxin operon include the post translational activator gene (C), the structural gene (A), and the B and D genes which encode proteins that are required for secretion of the activated Lkt toxin.
The term xe2x80x9cpartially or fully inactivatedxe2x80x9d as used herein the claims and description includes modification of a gene by recombinant DNA techniques including introduction and deletion of DNA from the gene including single or multiple nucleotide substitution, addition and/or deletion including full or partial deletion of the gene, using a target construct or plasmid segregation; and
chemical induced-, radiation induced- or site specific mutagenesis.
The present applicants have found that a precursor of Lkt toxin has reduced toxic activity. Surprisingly, the present applicants have also found that the Lkt toxin precursor is capable of inducing an immune response in an animal that offers cross protection against heterologous challenge with a microorganism which produces the Lkt toxin.
Accordingly, in a preferred embodiment of the invention the inactivated Lkt toxin is a precursor of Lkt toxin. The precursor may be an unprocessed expression product of the Lkt structural gene. The Lkt structural gene may be the Lkt A gene.
The microorganism may be one which does not naturally produce an Lkt toxin. The microorganism may be a bacterium, virus or fungus into which the Lkt structural gene, such as the Lkt A gene, has been introduced.
In a preferred embodiment, however, the microorganism is one which naturally produces an Lkt toxin. The microorganism which naturally produces an Lkt toxin may be Pasteurella haemolytica. 
The present applicants have found that a microorganism which naturally produces an Lkt toxin may be engineered to produce an inactive Lkt toxin precursor by eliminating the post-translational activator of the precursor product. Accordingly, in a preferred embodiment the microorganism is unable to produce a post-translational activator of the Lkt toxin precursor or produces an inactivated post-translational activator of the Lkt toxin precursor. The post-translational activator may be a product of the Lkt C gene.
In a preferred embodiment the Lkt C gene of the microorganism is inactivated or partially or fully deleted. The Lkt C gene may be inactivated by site specific mutagenesis. The Lkt C gene may be inactivated by any single or multiple nucleotide substitution, addition and/or deletion. Preferably, the Lkt C gene is inactivated by homologous recombination using a targeting construct. The targeting construct may include a selectable marker flanked by sequences homologous to sequences flanking the desired insertion site. The selectable marker may be a gene which confers resistance to a toxic substance such as mercury or may be an antibiotic resistance determinant. The antibiotic resistance determinant may be a gene coding for ampicillin resistance, kanamycin resistance or streptomycin resistance.
In some circumstances it may be undesirable to have a functional antibiotic resistance gene incorporated into a modified microorganism. Accordingly, the present invention contemplates a targeting construct which includes genetic elements, such as repeat sequences, which facilitate excision of the antibiotic resistance gene once the targeting construct has undergone homologous recombination with the host chromosome.
The present invention also contemplates a targeting construct which does not include a selectable marker. For example, the targeting construct may include a segment of the Lkt C gene which contains a deletion. Homologous recombination of the targeting construct with the host chromosome may result in the introduction of a deletion into the chromosomal Lkt C gene. Selection for recombinants may then be based on the absence of production of the Lkt toxin.
The targeting construct may be introduced directly into the host cell in a linear form. Alternatively, the targeting construct may be introduced via a suicide or non-replicating vector. The suicide vector may be any plasmid which does not replicate in the host microorganism. Microorganisms which naturally produce Lkt toxins are often non-permissive hosts for pEP vectors. Accordingly, pEP vectors are examples of suicide vectors which may be used in the present invention.
In another embodiment, site specific mutagenesis may be achieved by the technique of plasmid segregation. For example, a plasmid which contains a fragment of an Lkt C gene interrupted by a selectable marker gene may be introduced to a microorganism. The microorganism may be subsequently transformed with a second plasmid containing a second selectable marker gene. Host cells containing both plasmids may then be passaged through media which selects only for the second plasmid. Selection for the second plasmid may act against maintenance of the first plasmid. The first plasmid may, therefore, be lost, but in some cases recombination of the interrupted Lkt C gene fragment containing the selectable marker into the chromosome may occur. This process therefore may encourage recombination of the interrupted Lkt C gene into the chromosomal Lkt C gene, thus inactivating the chromosomal Lkt C gene.
In a further aspect of the present invention, there is provided an expression vector which encodes an Lkt toxin wherein said Lkt toxin is partially or fully inactivated, said vector encoding an Lkt toxin gene including an Lkt structural and/or post-translational activator gene wherein said Lkt toxin operon is partially or fully inactivated.
The term xe2x80x9cexpression vectorxe2x80x9d as used herein the claims and description includes a chromosomal or extrachromosomal element which is capable of expressing a DNA sequence including a foreign DNA sequence.
The Lkt A gene product may be expressed from a chromosomal Lkt A gene. The chromosomal Lkt A gene may be located in its natural position on the chromosome or may be inserted into the chromosome at a position other than its natural location. In addition, the Lkt gene product may be expressed from an Lkt A gene located on an extrachromosomal element such as a plasmid. In one embodiment, therefore, an extrachromosomal element containing an Lkt A gene may be introduced to a microorganism which has a functional chromosomal Lkt A gene and an inactivated chromosomal Lkt C gene. The Lkt A product expressed from the extrachromosomal element may supplement the Lkt A product expressed from the chromosomal gene.
Alternatively, the Lkt A gene product may be expressed entirely from an Lkt A gene or genes located on extrachromosomal elements such as plasmids. The Lkt A genes located on extrachromosomal elements may be expressed either in the presence or absence of selection for the extrachromosomal element. Thus, in one embodiment an extrachromosomal element containing an Lkt A gene may be introduced into a microorganism which lacks functional chromosomal Lkt A and Lkt C genes. The microorganism which lacks functional Lkt A and Lkt C genes may be produced by mutagenesis of the microorganism. The mutagenesis may result in deletion of the Lkt A and Lkt C genes or portions thereof.
The extrachromosomal element may be a recombinant expression vector which includes the Lkt A gene. Preferably the recombinant expression vector allows expression of the Lkt A gene in microorganisms which naturally produce Lkt toxins. The recombinant expression vector may allow expression of the Lkt A gene in P. haemolytica. The recombinant expression vector may be derived from a pIG plasmid. The recombinant plasmid may be derived from pIG3B. The recombinant plasmid may be pIG3B-Lkt.
Bacterial vector systems based on APP (Ph) provide an alternative means to deliver xe2x80x9cnaked DNAxe2x80x9d vaccine molecules to host cells. Such naked DNA vaccine/expression systems would include a plasmid capable of replicating in the bacterial system, and a eukaryotic promoter controlling the expression of the foreign/recombinant gene of interest.
In a preferred embodiment the microorganism is able to produce one or more functional proteins which facilitate secretion of Lkt toxin molecules. The microroganism may have functional Lkt B and/or Lkt D genes. In another embodiment, the microorganism is unable to produce at least one of the proteins involved in secretion of Lkt toxin molecules or produces at least one inactive protein involved in secretion of Lkt toxin molecules. The microorganism may have an inactive Lkt B and/or Lkt D gene. Thus, the microorganism may be unable to secrete active or inactive Lkt toxin molecules.
In another aspect the present invention provides a vaccine composition for inducing an immunological response in a host animal inoculated with said vaccine composition, said vaccine composition including an Lkt toxin precursor. The Lkt toxin precursor may be an unprocessed expression product of an Lkt structural gene. The Lkt structural gene may be an Lkt A gene.
The present invention further provides a vaccine composition for inducing an immunological response in a host animal inoculated with said vaccine composition, said vaccine composition including a modified microorganism which produces an Lkt toxin, wherein said Lkt toxin is partially or fully inactivated. Preferably the inactivated Lkt toxin is a precursor of an Lkt toxin. The precursor may be an unprocessed expression product of an Lkt structural gene. The Lkt structural gene may be an Lkt A gene. Preferably, the microorganism is Pasteurella haemolytica. Preferably the Lkt C gene of the microorganism is inactivated or deleted.
In a preferred embodiment the vaccine composition which includes a modified microorganism is a live vaccine.
A vaccine composition of the present invention may be incorporated in any pharmaceutically acceptable vehicle with or without added adjuvants or immunostimulatory molecules.
The adjuvant may be of any suitable type. The adjuvant may be selected from vegetable oils or emulsions thereof, surface active substances, e.g., hexadecylamine, octadecyl amino acid esters, octadecylamine, lysolecithin, dimethyl-dioctadecyl-ammonium bromide, N,N-dicoctadecyl-Nxe2x80x2-Nxe2x80x2 bis (2-hydroxyethyl-propane diamine), methoxyhexadecylglycerol, and pluronic polypols; polyamines, e.g., pyran, dextransulfate, poly IC, carbopol; peptides, e.g., muramyl dipeptide, dimethylglycine, tuftsin; immune stimulating complexes (ISCOMS); oil emulsions; and mineral gels and suspensions. A mineral suspension such as alum, i.e. aluminium hydroxide (Al(OH)3), aluminum phosphate or aluminium sulphate is preferred. The adjuvant may be present in amounts of from approximately 1 to 75% by weight, based on the total weight of the vaccine composition.
It will be appreciated that a vaccine according to the present invention, which includes an Lkt toxin precursor or a microorganism capable of producing an Lkt toxin precursor, has the potential to provide protection against a range of serovars of a microorganism which produces the corresponding Lkt toxin.
In another aspect the present invention provides a biological vector including a Pasteurella haemolytica bacterium, wherein said bacterium has been modified such that it is incapable of producing an active Lkt toxin.
In a preferred embodiment, the modified bacterium is unable to produce a post-translational activator of the Lkt toxin precursor or produces an inactivated post-translational activator of the Lkt toxin precursor. The post-translational activator may be a product of an Lkt C gene. In a preferred embodiment the Lkt C gene of the modified bacterium is inactivated or deleted. The Lkt C gene may be inactivated by any single or multiple nucleotide substitution, addition and/or deletion.
The Lkt C gene may be inactivated by the introduction of a targeting construct containing a selectable marker into the Lkt C chromosomal gene through site specific recombination. The targeting construct may include genetic elements, such as repeat units, which facilitate excision of the antibiotic resistance gene once the targeting construct has undergone homologous recombination with the host chromosome. Alternatively, a targeting construct which does not contain a selectable marker may be used to introduce a deletion in the Lkt C chromosomal gene.
The term xe2x80x9cbiological vectorxe2x80x9d is used in its widest sense to include a biological means suitable for expression of biologically active molecules. The biological means is preferably a viable microorganism although dead organisms could be employed. The biological vector may be non-pathogenic or rendered avirulent or may be given in non-pathogenic or avirulent effective amounts. The term xe2x80x9cbiologically active moleculesxe2x80x9d includes functional molecules such as growth factors, hormones, enzymes, antigens or antigenic parts thereof, cytokines such as interleukins, interferons and tumor necrosis factors. The molecules may be expressed naturally by the biological vector. Alternatively, the molecules may be recombinant molecules expressed by transforming the biological vector with a plasmid carrying a gene or genes encoding the biologically active molecule and which is then expressed; or where the plasmid and/or gene or genes and/or parts thereof are integrated into the host genome, which includes the chromosome and/or any naturally or non-naturally occurring extra-chromosomal element, wherein the gene or genes or parts thereof are expressed.
It will be appreciated that a biological vector of the present invention may be used to provide one or more useful proteins to the host animal. The proteins so provided may act in synergy to bring about an enhanced reaction in the host animal. For example, the biological vector may produce an antigen in combination with a molecule which enhances an immunogenic response in the host animal to the antigen. The molecule which enhances the immunogenic response may be a cytokine.
It will also be appreciated that a biological vector of the present invention may be used to provide a multivalent vaccine. The term xe2x80x9cmultivalent vaccinexe2x80x9d is used in its most general sense and extends to a modified microorganism capable of inducing an immune response to two or more distinct antigenic epitopes on or expressed by the modified microorganism where the two or more epitopes are indigenous to the modified microorganism. More commonly, however, a multivalent vaccine includea modified microorganism capable of inducing an immune response to virulent forms of said microorganism as well as to heterologous antigens expressed by said microorganism (such as recombinant antigens or those introduced by transduction, conjugation or transformation) and which are not indigenous to the microorganism. In this regard, a multivalent vaccine may be directed to two or more pathogenic agents. Preferred multivalent vaccines are those capable of inducing an immune response against an Lkt toxin and to at least one antigenic eptiope from one or more pathogenic agents. The pathogenic agents may be selected from bacterial pathogens such as Pasteurella spp., Haemophilus spp., Moraxella spp., Leptospira spp., Streptococcus spp., Salmonella spp., E. coli, Fusobacterium spp., Clostridium spp., Mycobactedum spp. The pathogenic agents may also be selected from endoparasites such as Haemonchus spp., Trichostrongylus spp., or ectoparasites such as Boophilus spp. Alternatively, the pathogenic agents may be selected from viral pathogens such as bovine viral diarrhoea virus (BVDV), parainfluenza virus (PI3), infectious bovine rhinotrachetis (IBR), coronavirus, rotavirus.
The present invention further provides a method of producing a modified organism which produces an Lkt toxin which is partially or fully inactivated which method includes
providing a microorganism which produces an active Lkt toxin; and
inactivating or deleting the Lkt C gene.
The present invention further provides a method of producing a modified organism which produces an Lkt toxin which is partially or fully inactivated which method includes
providing a microorganism which is incapable of producing an active Lkt toxin; and
introducing a functional Lkt A gene into said microorganism.
The invention in yet a further aspect provides a method for vaccinating an animal against an Lkt toxin producing microorganism, said method including administering to said animal an immunologically effective amount of a vaccine in accordance with the present invention.
The method of vaccination may be utilised in the treatment of production animals such as pigs, cattle, sheep, goats. The method of vaccination may also be used in the treatment of companion animals such as horses, dogs and cats. The method of vaccination may also be used in the treatment of humans. In a preferred embodiment the method of vaccination is utilized in the treatment of cattle. Preferably the method of vaccination is utilized in the treatment of bovine respiratory disease (BRD) complex.
Administration of a vaccine or vaccine vector in accordance with the present invention may be by any suitable route such as by oral or parenteral administration. The administration may be mucosal such as nasal or vaginal. Alternatively, administration may be intramuscular, intradermal, subcutaneous or intraperitoneal. The preparation may be in dry or liquid form. The route of administration chosen may also necessitate additional components such as protease inhibitors, anti-inflammatories and the like.
The invention in yet a further aspect provides a method for vaccinating an animal against a pathogenic organism, said method including administering to said animal an effective amount of a vaccine vector in accordance with the present invention wherein said vaccine vector synthesises an immunologically effective amount of an antigen of said pathogenic organism.
In yet another aspect the present invention provides a method for the production of an inactive Lkt toxin which method includes culturing a modified microorganism in accordance with the present invention and recovering the inactive toxin produced by said microorganism. The inactive Lkt toxin produced by this method may, for example, be used as the active immunogen in a vaccine for stimulating a protective immune response against an Lkt toxin.
Throughout the description and claims of this specification, the word xe2x80x9ccomprisexe2x80x9d and variations of the word, such as xe2x80x9ccomprisingxe2x80x9d and xe2x80x9ccomprisesxe2x80x9d, is not intended to exclude other additives, components, integers or steps.
In order that the invention may be more readily understood we provide the following non-limiting examples.